The members of the beta-globin family of genes are expressed sequentially during human development. The epsilon-globin gene is the first to be expressed, is actively transcribed until the sixth week of development, and is inactive later in development. Expression of the beta-like-globin genes is under the control of the beta-globin locus control region (LCR), encompassing erythroid specific DNase I hypersensitive sites (HS) 5-18 kilobases upstream of the epsilon-globin gene. The LCR has long range effects on the chromatin structure of the beta- globin locus, and it activates transcription of the different globin gene promoters as the genes are expressed during development. Using in vitro protein-DNA binding studies, clustered point mutations, and transient expression assays with a reporter gene, we have identified transcription factors that can be expected to participate, in vivo, in mediating the interaction of the LCR with the epsilon-globin gene promoter. These include the ubiquitous transcription factor Sp1 (or a related family member) and the erythroid transcription factors GATA-1 and NF-E2. To study the promoter-LCR interaction in the context of chromatin in vivo we have constructed a minichromosomal vector containing a marked epsilon- globin gene. Expression of the minichromosomal epsilon-globin gene in K562 human erythroid cells is dependent on the presence of the beta- globin LCR in cis. Regulated expression of the epsilon-globin gene on the minichromosomes offers a means to study at high resolution the effects of the LCR on the chromatin structure of the epsilon-globin gene, and the possibility to dissect the promoter-enhancer interaction of an activated gene assembled in vivo into chromatin.